J. Cell Set. 64, 137-146 (1983) 137 Printed in Great Britain © The Company of Biologists Limited 1983 INFECTION OF MACRONUCLEAR ANLAGEN OF PARAMECIUM CAUDATUM WITH THE MACRONUCLEUS-SPECIFIC SYMBIONT HOLOSPORA OBTUSA MASAHIRO FUJISHIMA Biological Institute, Faculty of Science, Yamaguchi University, Yamaguchi 753, Japan AND HANS-DIETER GORTZ Zoologisches Institut der Universitdt Munster, D-4400 Munster, Federal Republic of Germany SUMMARY The gram-negative bacterium Holospora obtusa is an endonuclear symbiont of Paramecium caudatum, which is incorporated into the host cells via the food vacuoles and infects their macronucleus exclusively, but never the micronucleus. Since these two kinds of nuclei originate from a fertilization nucleus, it is assumed that the macronucleus acquires a property necessary for it to be recognized by the bacterium at a certain time during the nuclear differentiation process. We found that this property is acquired by four of the eight postzygotic nuclei as soon as the four nuclei differentiate morphologically into the macronuclear anlagen. INTRODUCTION Ciliated protozoa contain two kinds of nuclei, which differ in ploidy, morphology, function and genetic content. The polyploid macronucleus has a high transcriptional activity (Gorovsky & Woodard, 1969; Ron & Urieli, 1977), whereas the diploid micronucleus has condensed, inactive chromatin compared to that of the macronucleus (Rao & Prescott, 1967; Pasternak, 1967) and functions as germ nucleus. Some micronuclear DNA sequences are not present in the macronucleus (Ammermann, Steinbriick, Berger & Henning, 1974; Lauth, Spear, Heumann & Prescott, 1976; McTavish & Sommerville, 1980; Yao, 1982). Despite the apparently different nature of these nuclei, however, both originate from a common fertilization nucleus. How the macro- and the micronucleus, of common genetic origin, differen- tiate into two morphologically and functionally different nuclei within a cell is un- resolved. In Paramecium caudatum, four species of bacterial endonuclear symbionts are known (Hafkine, 1890; Gortz, 1980): Holospora elegans, H. undulata, H. obtusa and ms-2. The former two inhabit the micronucleus exclusively, but never the macronucleus or cytoplasm. On the other hand, the latter two inhabit the macronucleus only. The Holospora species can easily infect the specific nucleus via the food vacuoles when a homogenate of symbiont-bearing cells is added to symbiont- free cells (Ossipov & Ivakhnyuk, 1972; Ossipov, Skoblo & Rautian, 1975; Gortz & 138 M. Fujishima and H.-D. Gortz Dieckmann, 1980; Gortz, 1980). Although the cause of the nuclear specificity of these bacteria has not been clarified, it is at least clear that they possess the ability to infect a specific nucleus, either the macro- or the micronucleus. This ability of the symbionts provides an opportunity to examine the stage at which the postzygotic nuclei differen- tiate into macro- and micronuclei, with respect to the acquisition of a property necess- ary for them to be recognized and infected by the symbionts. We demonstrate here, by using the macronucleus-specific symbiont//. obtusa, that the macronuclear anlagen acquire the property needed by the macronucleus for an infection with H. obtusa as soon as the postzygotic nuclei differentiate morphologi- cally into the macronuclear anlagen. The result suggests that this property is acquired when macronuclear differentiation begins. MATERIALS AND METHODS Strains and culture conditions The cells used in this study were Paramecium caudatitm syngen 3, mating type V, strain 27aG3, and mating type VI, strain 27aG3BC8-l. These strains were kindly supplied by Dr Y. Tsukii, Hosei University. The original Holospora obtusa-bearing strain C101 (syngen unknown) was collected in Munster, FRG. Later, the symbionts infected strain 27aG3 and this newly infected strain was used for obtaining the symbionts in this study. The culture medium used was 1 -25 % (w/v) fresh lettuce juice in Dryl's solution (Dryl, 1959) inoculated with a non-pathogenic strain of Klebsiella pneumoniae 1 day before use (Hiwatashi, 1968). In ordinary cultures several hundred cells were inoculated into 2 ml culture medium and then 4 ml, 10 ml and 10 ml of fresh medium were added on successive days. Cultures were kept at 25 °C. One or two days after the final feeding, mating reactivity increased to its maximum intensity. Infection Holospora species show two morphologically distinct forms in their life cycle (Ossipov & Ivakhnyuk, 1972; Ossipov et al. 1975; Gortz & Dieckmann, 1980): a reproductive short form and an infectious long form. The former is observed predominantly in nuclei of vegetatively growing host cells and the latter in that of starved cells. Only the long form is infectious. Infection of macronuclei or macronuclear anlagen of P. caudatum with H. obtusa was achieved as follows. Cultures of symbiont-bearing cells in stationary phase were strained through four layers of fine gauze to remove gross debris. Then the cells were harvested by centrifuging for 3 min at 1000 rev./min, and homogenized by hand in a Teflon homogenizer at 0—4CC. The density of the symbionts in the homogenate was counted with a blood-counting chamber and adjusted to 4 X lO'symbionts/ml by adding Dryl's solution. Recipient cells and the homogenate containing//, obtusa were mixed in depression slides at 1500 cells/ml and 4 X lO^ymbionts/ml, respectively, at 25 °C. Cytological methods for observation of nuclei and symbionts Cells infected with//, obtusa were harvested by centrifugation in a hand-operated centrifuge, air- dried on glass slides, and fixed in a mixture of acetic acid and ethanol (1:3, v/v) for 10 min at room temperature. Preparations were stained by the Feulgen reaction and counterstained with 0-25 % (w/v) fast green FCF. Observations were made using a differential interference-contrast microscope at a magnification of X1000. Temporal observation of the symbionts in host macronuclei was made as follows. Cells were pipetted with a few microlitres of culture medium onto glass slides and fixed in 4 % (w/v) OsO« vapour by inverting the slide over a small vial of the fixative for 3-4 s. The cells were then observed either unstained, or stained with aceto-orcein. Observation of food vacuoles Cell suspensions were mixed with a drop of Indian ink solution for. 15 min at a density of about Macronuclear differentiation in Paramecium 139 1500 cells/ml at 25 °C. Then the cells were fixed and stained by the Feulgen reaction and with fast green FCF as described before. The cells able to form food vacuoles are expected to ingest Indian ink and form many black-coloured food vacuoles. RESULTS Time-course of infection vnth H. obtusa In order to determine the time needed for infection of the macronucleus of P. caudatum with H. obtusa, symbiont-free cells were mixed with the homogenate of symbiont-bearing cells at 25°C (see Materials and Methods). Then the cells were fixed every 10 min for the first 2 h after mixing, and stained as described. The results are shown in Fig. 1. Ten minutes after mixing the cells with the symbiont-containing homogenate, the macronucleus was infected with//, obtusa in about 20 % of the cells. The symbionts were also observed in the cytoplasm, some were in food vacuoles and some apparently outside the food vacuoles. The proportion of cells bearing symbionts in their macronuclei rose to about 60 % at 30 min after mixing and to about 100 % at 60 min. Mean numbers of the symbionts observed in the individual macronuclei at 10, 30 and 60 min after mixing were about 1, 2 and 3, respectively. The symbionts were never observed in the micronucleus. The data show that the infection of the macronucleus with H. obtusa begins remarkably quickly after mixing. Since the symbionts can be observed not only near the macronucleus but also near the 100 10 90 80 70 u 60 I 50 5.l 8 40 cel 5 30 a? "5 20 6 c 10 c <0 0 10 20 30 40 50 60 120 Time after mixing with H. obtusa (min) Fig. 1. Time-course of infection of the macronucleus of P. caudatum with Holospora obtusa. H. obtusa-iTee cells were mixed with an homogenate of the symbiont-bearing cells (see Materials and Methods). At each time-point, a hundred cells were observed, and the ratios of cells that had symbionts in the macronucleus (• •) and the mean numbers of the symbionta per macronucleus (O O) were plotted. C.L. is confidence limit. 140 M. Fujishima and H.-D. Gortz micronucleus, it is evident that the symbionts can infect the macronucleus but not the micronucleus. Similar observations were reported by Ossipov and colleagues (see Ossipov, 1981, for a review). Infection of macronuclear anlagen of exconjugant cells with H. obtusa As shown in previous studies (Calkins & Cull, 1907; Klitke, 1916; Saito & Sato, 1961; Mikami & Hiwatashi, 1975; Mikami, 1980), in P. caudatum the synkaryon (fertilization nucleus) normally divides three times successively; four of the resultant nuclei become macronuclear anlagen, one becomes a micronucleus, and the remain- ing three degenerate. The old macronucleus is transformed into a loosely wound skein (skein formation) at about the time of the third (last) postzygotic division and subsequently breaks into many fragments (macronuclear fragmentation). Recently, it has been found by Mikami (1980) that the determination of the macro- and the micronuclear differentiation occurs immediately after the third nuclear division and is closely related to a brief localization of the nuclei at the opposite ends of the cell, with the prospective macronuclei in the posterior region and the prospective micronuclei in the anterior region. Similar results were also reported in P. tetraurelia (formerly called syngen 4 of P. aurelia; Sonneborn, 1975) (Grandchamp & Beisson, 1981). Hereafter, this stage will be tentatively called the 'determination stage for nuclear differentiation'. The earliest morphological change of the nuclei after the determination stage is the appearance of heterochromatic aggregates in the four macronuclear anlagen, which is followed by increased stainability with fast green.